運用CRISPR活化系統用於調控脂肪幹細胞內長鏈非編碼核糖核酸及改促進骨再生

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研究生：	阮氏翹娘 Thi Kieu Nuong Nguyen
論文名稱：	運用CRISPR活化系統用於調控脂肪幹細胞內長鏈非編碼核糖核酸及改促進骨再生 CRISPR Activation of Long Non-coding RNA in Mesenchymal Stem Cell for Bone Regeneration
指導教授：	胡育誠 Hu, Yu-Chen
口試委員:	宋信文 Sung, Hsing-Wen 陳韻晶 Chen, Yunching 張毓翰 Chang, Yu-Han 林進裕 Lin, Chin-Yu
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	111
中文關鍵詞：	進骨再生
外文關鍵詞：	Bone healing, CRISPRa, DANCR, H19, dCas9-VPR, Split dCas12a, lncRNA
相關次數：	點閱：4 下載：0
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大範圍的骨缺陷在目前臨床骨科醫學依然是一個難題。儘管移植能分化至軟骨組織的ASC 和BMSC可以增進修復，但是這種以細胞為基礎的療法距離臨床仍有一大步，因此需要研發更新的方法來促進幹細胞分化，應用在大範圍骨缺陷的治療。近年來，長鏈非編碼RNA (lncRNAs)已經被視為調控基因的重要因子，lncRNAs帶有超過200個核甘酸的並且可以被RNA polymerase II轉錄，但不會轉譯，而lncRNAs在細胞分裂與分化扮演著多種的角色。DANCR和H19這兩個lncRNAs被發現可以促進軟骨再生，但是目前並沒有應用在幹細胞軟骨修復的研究，且機制並未明了。
CRISPR activation (CRISPRa)是一個非常好建構的系統並且可以啟動細胞的內源基因表現，讓我們可以對於在細胞核內的lncRNA進行調控，這能突破以往無法直接過表現lncRNA的瓶頸。在這篇研究當中，我們建構兩套CRISPR系統分別在rASC和BMSC啟動DANCR 和H19。於第一套系統中，我們建構出不同的桿狀病毒載體來表現不同的dCas9，並分別連接三個複合啟動子包含VP64, p65 和 Rta (SadCas9-VPR, SpdCas9-VPR, St1dCas9-VPR, NmCas9-VPR)，我們發現在四種不同的dCas9中，SadCas9-VPR 能最有效的在rASC啟動DANCR。在第二套系統中，我們建構的Split dCas12a將dCas12a 分成N片段和C片段，兩片段分別連接兩個p65-HSF1啟動子，經過表現後N片段和C片段能夠自動組成一個帶有四個啟動子的全長dCas12a來啟動rBMSC的H19。我們更進一步的發現DANCR和H19在兩套CRISPR的啟動調控下，能夠刺激一些往軟骨組織分化的相關基因的表現，如:Sox9, Col2a1和Acan。另一方面，我們進一步的利用GAG和II型膠原蛋白檢測證實軟骨能在3D骨架形成。最重要的是，我們將基因調控過的rASCS和rBMSC移植進入SD大鼠後，能夠促進頭蓋骨的修復。綜合而言，我們的成果展現出我們的兩套CRISPR系統能夠調控lncRNAs來誘導rASC和BMSC往軟骨組織分化，並且提供一個有潛力的療法應用在骨修復。

Healing of large calvarial bone defects in adults remains to be a challenging task. Although implantation of adipose-derived stem cell (ASC) or bone marrow stem cell (BMSC) that differentiate towards chondrogenic lineage improves the healing process, this cell-based therapy is still far from perfect, thus requiring new methods to stimulate cell differentiation so as to further augment calvarial bone healing. In recent years, long non-coding RNAs (lncRNAs) have been recognized as essential elements of gene regulation. lncRNAs with over 200 nucleotides in length are transcribed by RNA polymerase II, but are not translated. lncRNAs regulate various cellular processes such as cell proliferation development and differentiation via diverse mechanisms. lncRNA Differentiation Antagonizing Non‑protein Coding RNA (DANCR) and lncRNA H19 (H19) were exposed to enhance chondrogenic differentiation, but its function in mesenchymal stem cells and bone regeneration have yet been explored.
Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) is easy to construct and may activate lncRNA expression from the endogenous genomic locus, allowing us to carry out the function of lncRNAs which are located in nucleus and that cannot be readily evaluated by traditional overexpression method. In this study, we constructed two CRISPRa systems to activate DANCR and H19 expression in rASC and BMSC, respectively. Firstly, we constructed different baculovirus vectors to express different dCas9 orthologues by fusing a tripartite transcription activator domain consisting of VP64, p65 and Rta (SadCas9-VPR, SpdCas9-VPR, St1dCas9-VPR, NmCas9-VPR) and compared the efficient activation of DANCR using the 4 different dCas9-VPR. We found that among the 4 different dCas9-VPR systems, SadCas9-VPR is more efficient activation of DANCR in rat ASC (rASC). Secondly, we constructed the Split dCas12a system where dCas12a is expressed as N domain and C domains, each of which is fused with two transcription activators consisting of P65 and HSF1(P65- HSF1). After expression, the split N and C domains spontaneously assemble to become a full-length dCas12a with 4 synthetic transcription activators, resulting in potentiating H19 activation effects in rat BMSC (rBMSC). Furthermore, we showed that CRISPR-mediated upregulation of DANCR and H19 stimulated the expression of chondrogenic differentiation markers such as SRY-box transcription factor 9 (Sox9), collagen type II alpha 1 chain (Col2a1) and aggrecan (Acan). On the order hand, the histological analysis as well as Glycosaminoglycans (GAG) and collagen type 2 (Col II) content quantification further confirmed the cartilage formation in three-dimensional (3D) culture. Importantly, implantation of the engineered rASCs and rBMSC into Sprague Dawley (SD) rats considerable promoted calvarial bone healing after transplantation. Taken together, our results demonstrated that CRISPR-mediated lncRNAs drives rASC and BMSC towards chondrogenesis lineage and offer a potential therapeutic target for bone regeneration.

Table of content
Acknowledgment    I
Abstract        III
Table of content    VI
List of Figures    IX
List of Table    XI
Chapter 1.    Literature review    1
1.1. Bone tissue and healing process    1
1.1.1. Structure of bone tissue    1
1.1.2. The process of bone healing    3
1.1.3. Development in bone tissue engineering    5
1.2. Gene therapy in bone regeneration    7
1.3. Baculovirus expression system for gene delivery    9
1.4. CRISPR activation system    12
1.5. Function and mechanism of long non-coding RNA    15
1.6. Research objective    16
Chapter 2.    Experimental Method    28
2.1. Construction and preparation of baculovirus vectors    28
2.1.1. Construction of CRISPR-VPR system    28
2.1.2. Construction of sgRNAs for CRISPR-VPR system    28
2.1.3. Construction of Split dCas12a system    29
2.1.4. Construction of crRNAs for split dCas12a system    30
2.1.5. Bacmid generation (Bac to Bac system)    31
2.1.6. Bacmid purification    31
2.1.7. P0 virus production    31
2.1.8. Amplification of baculovirus    32
2.2. Cell isolation and culture    32
2.2.1. Rat adipose-derived stem cell (rASC)    32
2.2.2. Rat bone marrow stem cell (rBMSC)    33
2.3. Baculoviral transduction    34
2.4. Hek293 transfection    34
2.5. Electroporation    35
2.6. Luciferase reporter assay    35
2.7. Real-time quantitative polymerase chain reaction (RT-qPCR)    36
2.8. Differentiation induction    36
2.9. Alcian blue staining, Safranin O staining and Oil Red O staining    36
2.10. Western blotting    37
2.11. Qualitative and quantitative analysis cell/scaffold constructs    38
2.12. Surgical procedures of animal experiment    39
2.13. Bone regeneration analysis    39
2.14. Histological and immunohistochemical staining    39
2.15. Statistical analysis    40
Chapter 3.    Result and discussion    48
3.1. Overexpression of DANCR promotes chondrogenic differentiation    48
3.2. Validation of CRISPRa-VPR system for DANCR activation in mesenchymal stem cells    49
3.3. CRISPRa-VPR stimulates chondrogenesis by DANCR activation    50
3.4. CRISPRa-VPR-mediated DANCR activation stimulates ASCs chondrogenesis and cartilage formation    50
3.5. DANCR activation by CRISPRa promotes calvarial bone healing    51
3.6. DANCR promotes chondrogenic differentiation by upregulating Smad3 via sponging miR-214-3p and miR-203-3p    52
3.7. Discussion    54
Chapter 4.    Result and discussion (II)    72
4.1. Validation of split dCas12a system in Hek293 cells    72
4.2. Screening the best target sites for lncRNA H19 activation in rASC using electroporation    73
4.3. Validation of split dCas12a system for H19 activation in mesenchymal stem cells using baculovirus    74
4.4. H19 activation by Split dCas12a stimulates rBMSC chondrogenesis    75
4.5. H19 activation by Split dCas12a inhibited adipogenic differentiation    77
4.6. Enhancement of cartilage formation in 3D cell/gelatin culture.    78
4.7. H19 activation stimulates BMSC to promote calvarial bone formation.    79
4.8. Discussion    80
Reference    98
 
List of Figures
Figure 1.1. The structure of long bone (A) and flat bone (B).    19
Figure 1.2. Classification of Bone.    20
Figure 1.3. Four types of cells in bone tissue    21
Figure 1.4. The stages of endochondral bone formation    22
Figure 1.5. The stages of intramembranous bone formation    23
Figure 1.6. The schematic outlines of the bone remodeling cycle and the balance of bone resorption and bone formation [3].    24
Figure 1.7. Structure of two types of baculovirus    25
Figure 1.8. Overview of adaptive immunity by CRISPR-Cas systems [77]    26
Figure 1.9. The functions of lncRNA [78]    27
Figure 2.1. Surgical procedures of animal experiment    47
Figure 3.1. Overexpression of DANCR promotes chondrogenic differentiation.    60
Figure 3.2. CRISPRa -VPR systems activate DANCR expression.    61
Figure 3.3. DANCR activation stimulate ASC chondrogenesis.    63
Figure 3.4. Engineered ASCs by CRISPRa stimulate chondrogenesis and cartilage regeneration.    64
Figure 3.5. μCT analysis of in vivo calvarial bone repair and quantitative analysis of bone healing.    66
Figure 3.6. Confirmation of bone healing by histological and immunohistochemical analysis.    67
Figure 3.7. DANCR upregulates Smad3 by competing for miR-214and miR-203a binding.    68
Figure 3.8. miR-214 and miR-203a bind directly to Smad3 mRNA.    70
Figure 3.9. Proposed mechanism of DANCR activation on chondrogenic differentiation.    71
Figure 4.1. Characterization of split dCas12a activator.    86
Figure 4.2. Screening the best target sites for H19 activation in rASC using electroporation.    87
Figure 4.3. Split dCas12a system activates H19 expression.    89
Figure 4.4. BMSC chondrogenesis was stimulated  by H19 activation.    91
Figure 4.5. H19 activation by Split dCas12a inhibited adipogenesis.    93
Figure 4.6. Effects of Split dCas12a transduction on the BMSC chondrogenic differentiation and cartilage regeneration.    94
Figure 4.7. μCT analysis of in vivo calvarial bone repair and quantitative analysis of bone healing.    96

 
List of Table
Table 2.1. Spacer (targeting) sequence for DANCR    41
Table 2.2. Spacer (targeting) sequence for ASCL1, HBG1 and IL1RN and H19    42
Table 2.3. Luria agar dish formula    43
Table 2.4. Sequences of Smad3-wt1, Smad3-mut1, Smad3-wt2 and Smad3-mut2    44
Table 2.5. Sequences of Smad3-wt1, Smad3-mut1, Smad3-wt2 and Smad3-mut2    45
Table 2.6. Primer sequences used for qRT-PCR    46


                                

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